Compositions and methods for preventing and treating cancer via modulating UBE1L, ISG15 and/or UBP43

ABSTRACT

Compositions and methods of using compositions that induce UBE1L or a ubiquitin-like protein ISG15, or inhibit a deconjugase UBP43 to degrade oncogenic proteins and enhance apoptosis of cancer (neoplastic) or pre-cancerous (pre-neoplastic) cells are provided. Methods for the prevention or treatment of cancer via administration of these compositions are also provided.

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/506,226, filed Dec. 29, 2004, which claims benefit ofPCT/US2003/006905, filed Mar. 5, 2003, which claims the benefit of U.S.Provisional Application No. 60/361,830, filed Mar. 5, 2002, the contentsof which are incorporated herein by reference in their entireties.

This invention was made with government support under Grant Nos.RO-1-CA62275 and RO-1-CA87546 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Acute promyelocytic leukemia (APL) (FAB M3) cases express the oncogenicproduct of the t(15; 17) chromosomal rearrangement, promyelocyticleukemia (PML)/retinoic acid receptor α (RAR α) (Nason-Burchenal, et al.(1996) In Molecular Biology of Cancer, ed. Bertino, Academic San Diego,1st Ed., pp 1547-1560; Nason-Burchenal & Dmitrovsky (1999) In Retinoids:The Biochemical and Molecular Basis of Vitamin A and Retinoid Action,eds. Nau & Blaneer, Springer, Berlin, pp. 301-322). All-trans-retinoicacid (RA) treatment causes complete remissions in these APL casesthrough induction of leukemic cell differentiation (Nason-Burchenal etal. (1996) supra; Nason-Burchenal & Dmitrovsky (1999) supra). A hallmarkof RA response in APL is PML/RARα degradation that reverses PML/RARαoncogenic effects (Kakizuka, et al. (1991) Cell 68:663-674; Yoshida, etal. (1996) Cancer Res. 56:2945-2948; Raelson, et al. (1996) Blood88:2826-2832; Nervi, et al. (1998) Blood 92:2244-2251; Zhu, et al.(1999) Proc. Natl. Acad. Sci. USA 96:14807-14812). Proteasomalinhibitors prevent PML/RARα proteolysis, despite RA treatment, which isindicative of a proteasome-dependent pathway in this degradation(Yoshida, et al. (1996) supra; Raelson, et al. (1996) supra; Nervi etal. (1998) supra; Zhu, et al. (1999) supra). PML/RARα expression resultsin dominant-negative transcriptional repression (Kakizuka, et al. (1991)supra; de The, et al. (1991) Cell 68:675-684). This repression isantagonized by pharmacological RA dosages that overcome inhibitoryeffects on transcription of the N-Cor/SMRT corepressor complex that hashistone deacetylase activity (Lin, et al. (1998) Nature (London)391:811-814; Grignani, et al. (1998) Nature (London) 391:815-818). RAtreatment recruits a coactivator complex that stimulates transcription,resulting in activation of target genes (Lin, et al. (1998) supra;Grignani, et al. (1998) supra).

To understand the molecular basis of RA response in APL, RA target geneidentification has been sought. GOS2 has been suggested as a putative RAtarget gene as determined by microarray analysis of APL cells (Tamayo,et al. (1999) Proc. Natl. Acad. Sci. USA 96:2907-2912). The precisefunction of GOS2 is not yet known, but it was first identified asregulated during the cell cycle (Russell & Forsdyke (1991) DNA CellBiol. 10:581-591), suggesting a role in cell cycle control.

Another candidate retinoid target gene is the CCAAT/enhancer bindingprotein epsilon (C/EBP epsilon) that contributes to retinoidtranscriptional effects in APL (Park, et al. (1999) J. Clin. Invest.103:1399-1408). However, this species has not been linked to thedegradation of PML/RARα.

Recent microarray analysis of RA-treated NB4 APL cells reported theprominent induction of UBE1L (ubiquitin-activating enzyme E1-like)(Tamayo, et al. (1999) supra). The proteasome-dependent degradation ofPML/RARα has also been proposed as a mechanism by which RA overcomesPML/RARα oncogenic effects (Yoshida, et al. (1996) supra; Raelson, etal. (1996) supra; Nervi, et al. (1998) supra; Zhu, et al. (1999) supra).Hammerhead ribozymes that target PML/RARα have been used to show howPML/RARα degradation signals apoptosis but not differentiation intransfected APL cells that are either RA-sensitive or RA-resistant(Nason-Burchanel, et al. (1998) Blood 92:1758-1767; Nason-Burchanel, etal. (1998) Oncogene 17:1759-1768).

SUMMARY OF THE INVENTION

The present invention features a method for identifying agents aspotential therapeutics against cancer by determining the ability of theagent to induce UBE1L and/or ubiquitin proteins such as ISG15 ordetermining the ability of the agent to inhibit the deconjugase UBP43.

The present invention also features a method for enhancing pro-apoptoticand degradative pathways of neoplastic (cancerous) cells orpre-neoplastic (pre-cancerous) cells with an agent that induces UBE1Land/or ubiquitin-like proteins such as ISG15 or an agent which inhibitsthe deconjugase UBP43.

Another feature of the present invention is to provide a method forpreventing or treating cancer in a patient which comprises administeringto a patient an agent which induces UBE1L and/or ubiquitin-like proteinssuch as ISG15 or an agent which inhibits UBP43.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of UBE1L or UBP43 cotransfection onUBE1L-mediated inhibition of cyclin D1. UBP43 antagonized UBE1L-mediatedinhibition of cyclin D1 while having no appreciable effect on actinexpression. Quantification of signals is provided.

FIG. 2 shows the effects of bexarotene treatment on clonal growth ofBEAS-2B human bronchial epithelial cells. Dose-dependent inhibition ofBEAS-2B cell growth by bexarotene was found.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that UBE1L and the UBE1L-dependent pathwaysuppress cancer growth. For example, UBE1L is the retinoid target genein APL that antagonizes PML/RARα oncogenic effects by triggeringPML/RARα degradation. The consequence of this action is the promotion ofapoptosis resulting in anti-oncogenic effects of UBE1L in APL and otherneoplastic or pre-neoplastic cell contexts, including lung cancer.Accordingly, compositions which target UBE1L or UBE1L-dependent pathwayproteins such as the ubiquitin-like protein ISG15 or the deconjugaseUBP43, find application in preventing and treating cancer.

Thus, the present invention features anticancer agents, methods fordesigning or screening for new anticancer agents and methods for usingsuch agents in the prevention or treatment of cancer. In one embodiment,agents of the invention induce the activity and/or expressionubiquitin-activating enzyme E1-like protein (UBE1L). In anotherembodiment, agents of the invention induce the activity and/orexpression of ubiquitin-like proteins such as ISG15. In an alternativeembodiment, agents of the invention inhibit the activity and/orexpression of the deconjugase UBP43. By inducing the expression and/oractivity of UBE1L or ISG15, or inhibiting the expression and/or activityof UBP43, pro-apoptotic and degradative pathways of neoplastic cells orpre-neoplastic cells are activated. In this regard, agents of thepresent invention are useful in preventing or treating cancerous orneoplastic as well as pre-cancerous or pre-neoplastic cells.

UBE1L is widely expressed in diverse human tissues and tumor cell lines.UBE1L acts as the activating enzyme for the ubiquitin-like proteinISG15. As demonstrated herein, UBE1L is a RA-inducible gene target inacute promyelocytic leukemia and U937 and THP1 cells, implicating abroad biological role for UBE1L. Expression of ISG15 as well as thedeconjugase UBP43 have also been found to be induced by RA and appear tobe regulated in a coordinated fashion with UBE1L. As shown herein, thisinduction occurs only in RA-sensitive cells, and not in RA-insensitivecells. Further, coordinate regulation and physical association of UBE1Land ISG15 induces degradation of oncogenic proteins including, but notlimited to, PML/RARα, cyclin D1, and PML, in RA-sensitive cells. Inaddition, the induction of degradation of these oncogenic proteins byUBE1L and ISG15 appears to preferentially trigger apoptosis. PML/RARαdegradation is inhibited by UBP43 transfection.

Similar analysis was conducted in lung cancer cells. In this analysis,UBE1L transduction was found to suppress cyclin D1 expression and growthof HBE and lung cancer cells. Transfection of the UBE1L-ISG15deconjugase, ubiquitin specific protein 18 (UBP43), antagonizedUBE1L-dependent inhibition of cyclin D1 and ISG15-cyclin D1 conjugation.In contrast, UBE1L knockdown increased cyclin D1 expression. Moreover,UBE1L conferred growth suppression by preferentially targeting cyclinD1. These findings demonstrate the role of UBE1L and UBE1L-dependentpathway proteins (i.e., ISG15 and UBP43) in growth suppression bytargeting cyclin D1 for repression.

Accordingly, agents that induce activity and/or expression of UBE1L orubiquitin-like proteins such as ISG15 and agents that inhibit activityand/or expression of the deconjugase UBP43 are expected to be useful intreatment of neoplasia and pre-neoplasia, particularly RA-sensitivecancers and cancers expressing oncogenic proteins such PML/RARα. Cancersof particular relevance include, breast cancer, APL, lung cancer, T-celllymphoma, ovarian cancer, gastric cancer.

Agents that selectively induce UBE1L or ISG15 expression or activity, orinhibit the expression or activity of UBP43 in APL are expected to causeanti-leukemic effects by triggering PML/RARα degradation and apoptosis.Based on findings presented here and previous reports (Yuan & Krug(2001) EMBO J. 20:362-371), UBE1L is also expected to have an importantbiological role beyond APL. UBE1L maps to chromosome 3p, a regionfrequently deleted in lung cancers; UBE1L repression is frequent in lungcancers (Kok, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6071-6075;Carritt, et al. (1992) Cancer Res. 52:1536-1541), where it may exert atumor suppressive effect. When coupled with the expression pattern ofUBE1L in human tissues or tumor cells (McLaughlin, et al. (2000) Int. J.Cancer 85:871-876) and the results reported herein, UBE1L and theUBE1L-dependent pathway (i.e., ISG15 and UBP43) regulate growth of bothnormal and neoplastic or pre-neoplastic cells.

Cancer cells, and in particular RA-sensitive cancer cells or cancercells expressing oncogenic proteins such as PML/RARα can be contactedwith agents of the present invention that induce UBE1L or ISG15expression or activity, or inhibit the expression or activity of thedeconjugase UBP43 to trigger degradation of oncogenic proteins such asPML/RARα and apoptosis in the cancer cells. Activation of this newlyidentified pathway is also expected to trigger degradation of otheroncogenic proteins in non-leukemia, including other neoplastic orpre-neoplastic cells such as lung and breast cancer cells. In particularembodiments, agents of this invention selectively induce UBE1L or ISG15activity or expression, or selectively inhibit UBP43 activity orexpression.

As used herein, a selective agent is any molecular species that is anUBE1L or ISG15 activator or UBP43 inhibitor but which fails to activateor inhibit, or activates or inhibits to a substantially lesser degreethe expression or activity of other any other protein in the cell. Inthis regard, selectivity of agents of the invention should alleviateundesirable clinical toxicities that complicate RA or arsenic trioxidetreatments. Methods for assessing the selectively of agents are known inthe art and can be based upon any conventional assay including, but notlimited to the determination of the half maximal (50%) inhibitoryconcentration (IC) of a substance (i.e., 50% IC, or IC₅₀), the bindingaffinity of the molecule, and/or the half maximal effectiveconcentration (EC₅₀). UBE1L, ISG15 and UBP43 nucleic acids and proteinsthat can be employed in such assays are well-known in the art andrespectively set forth, e.g., in GENBANK Accession Nos. NM_(—)003335 andNP_(—)003326 (human UBE1L or ubiquitin-like modifier activating enzyme7, UBA7); NM_(—)005101 and NP_(—)005092 (human ISG15); and NM_(—)017414and NP_(—)059110 (human UBP43 or ubiquitin specific peptidase 18,USP18).

Selective activators of UBE1L or ISG15 expression or activity include,but are not limited to nucleic acids encoding UBE1L (e.g., GENBANKAccession No. NM_(—)003335) or ISG15 (e.g., GENBANK Accession No.NM_(—)005101). These nucleic acid molecules can be used as is (e.g., asnaked DNA) or via vectors (e.g., a plasmid or viral vector such as anadenoviral, lentiviral, retroviral, adeno-associated viral vector or thelike) harboring nucleic acids encoding the UBE1L and/or ISG15.Desirably, a vector used in accordance with the invention provides allthe necessary control sequences to facilitate expression of UBE1L and/orISG15. Such expression control sequences can include but are not limitedto promoter sequences, enhancer sequences, etc. Such expression controlsequences, vectors and the like are well-known and routinely employed bythose skilled in the art.

Selective inhibitors of UBP43 expression include, but are not limitedto, agents such as microRNA, shRNA, siRNA, antisense, or ribozymemolecules specifically targeted to a nucleic acid molecule encodingUBP43 (e.g., GENBANK Accession No. NM_(—)017414). Such agents can bedesigned based upon routine guidelines well-known to those skilled inthe art. For example, siRNA target sites in a gene of interest can be19-27 nucleotides in length, include an AA dinucleotide sequence at the5′ end and preferably have a G/C content of 30-50% (see, e.g., Elbashir,et al. (2001) Nature 411: 494-498). Kits for production of dsRNA for usein RNAi are available commercially, e.g., from New England Biolabs, Inc.and Ambion Inc. (Austin, Tex., USA). Methods of transfection of dsRNA orplasmids engineered to make dsRNA are routine in the art. An exemplarysiRNA targeting human UBP43 is, e.g., 5′-gat ccc cag gag aag cat tgt tttcaa att caa gag att tga aaa caa tgc ttc tcc ttt ttt a-3′ (SEQ ID NO:1)and 5′-agc tta aaa aag gag aag cat tgt ttt caa atc tot tga att tga aaacaa tgc ttc tcc tgg g-3′ (SEQ ID NO:2)(Malakhova, et al. (2006) EMBO J.25(11): 2358-2367).

Selective inhibitors of UBP43 activity can be based upon the UBP43substrate Leu-Arg-Gly-Gly-Met-His-Ile-Ser (SEQ ID NO:3) (Malakhov, etal. (2002) J. Biol. Chem. 277:9976-9981). See, e.g., Kim ((1999)Biopolymeers (Peptide Science) 51:3-8) who teaches the design ofprotease inhibitors on the basis of substrate stereospecificity. Otherselective inhibitors can be derived from ubiquitin aldehyde, aninhibitor of ubiquitin-specific processing protease (UBP) family ofdeubiquitinating enzymes (Hu, et al. (2002) Cell 111:1041-1054), orN-ethylmaleimide, a general inhibitor of cysteine proteinases.

In addition to the above-referenced activators and inhibitors, it iscontemplated that any conventional screening assay can be employed foridentifying or selecting additional or more selective activators orinhibitors, or derivatives or analogs of known activators or inhibitorsfor UBE1L, ISG15, or UBP43. Screening, in accordance with the presentinvention, involves determining an agent's ability to induce UBE1L orISG15 expression or activity, or to inhibit UBP43 expression oractivity. An increase or decrease in the expression of a protein of thisinvention can be determined by contacting a cell expressing said proteinwith a test agent, and measuring whether the agent increases ordecreases the amount of mRNA encoding the protein (e.g., by northernblot analysis or RT-PCR) or amount of protein (e.g., by western blot ordot blot analysis) in the cell. An increase or decrease in the activityof a protein of this invention can be determined by contacting theprotein or a cell expressing said protein with a test agent, andmeasuring whether the agent increases or decreases the activity of theprotein (e.g., in an in vitro assay or based upon the expression of adownstream protein). For example, it can be determined whether cyclin D1expression is altered by the agent. By way of further illustration, anin vitro protease assay can be conducted with UBP43 and a substratedisclosed herein. Agents which increase the level of UBE1L or ISG15 mRNAor protein in the presence of a test agent as compared to UBE1L or ISG15mRNA or protein levels in the absence of the test agent is indicative ofthe test agent inducing UBE1L or ISG15 and being potentially useful asan anticancer agent. Similarly, agents that inhibit the expression ordeconjugase activity of UBP43 as compared to UBP43 expression oractivity in the absence of the test agent is indicative of a test agentinhibiting UBP43 and being potentially useful as an anticancer agent.Such agents can then be administered to prevent and treat cancer.

Agents can be identified and obtained from libraries of compoundscontaining pure agents or collections of agent mixtures. Examples ofpure agents include, but are not limited to, proteins, peptides, nucleicacids, oligonucleotides, carbohydrates, lipids, synthetic orsemisynthetic chemicals, and purified natural products. Examples ofagent mixtures include, but are not limited to, extracts of prokaryoticor eukaryotic cells and tissues, as well as fermentation broths and cellor tissue culture supernates. In the case of agent mixtures, one may notonly identify those crude mixtures that possess the desired activity,but also monitor purification of the active component from the mixturefor characterization and development as a therapeutic drug. Inparticular, the mixture so identified may be sequentially fractionatedby methods commonly known to those skilled in the art which may include,but are not limited to, precipitation, centrifugation, filtration,ultrafiltration, selective digestion, extraction, chromatography,electrophoresis or complex formation. Each resulting subfraction may beassayed for the desired activity using the original assay until a pure,biologically active agent is obtained. An exemplary library of cysteinepeptidase inhibitors to screen and identify UBP43 inhibitors aredisclosed by Abato et al. ((1999) J. Med. Chem. 42(19):4001-9). Theseinhibitors are based upon a cyclohexanone nucleus and are designed toprobe binding interactions in the S2 and S2′ binding sites. Moreover,Damarcus et al. ((2001) J. Org. Chem. 66(3):697-706) teach a smalllibrary of epoxy peptidomimetics as time-dependent reversible inhibitorsof cysteine proteases.

Library screening can be performed in any format that allows rapidpreparation and processing of multiple reactions such as in, forexample, multi-well plates of the 96-well variety. Stock solutions ofthe agents as well as assay components are prepared manually and allsubsequent pipetting, diluting, mixing, washing, incubating, samplereadout and data collecting is done using commercially available roboticpipetting equipment, automated work stations, and analytical instrumentsfor detecting the signal generated by the assay. Examples of suchdetectors include, but are not limited to, luminometers,spectrophotometers, calorimeters, and fluorimeters, and devices thatmeasure the decay of radioisotopes. It is contemplated that any suitableassay can be used in such screening assays. For example, activity ofUBP43 can be assessed with a purified peptide substrate or full-lengthISG15.

As indicated, selective agents of this invention find application inmethods for enhancing pro-apoptotic and degradative pathways ofneoplastic or pre-neoplastic cells and preventing or treating cancer, inparticular cancers such as APL or lung cancer. Generally, such methodsinvolve administering to a subject in need of treatment an agent thatselectively activates the expression or activity of UBE1L or ISG15, orselectively inhibits the expression or activity of UBP43 in an amountthat effectively reduces the expression of cyclin D1 or cancer cellproliferation by at least 60%, 70%, 80%, 90%, 95%, 99% or 100%. Subjectsbenefiting from treatment with an agent of the invention includesubjects confirmed as having cancer, subjects suspected of havingcancer, or subjects at risk of having cancer (e.g., subjects with afamily history or having been exposed to cancer-causing agents). In thecontext of this invention, a subject can be any mammal including human,companion animals (e.g., dogs or cats), livestock (e.g., cows, sheep,pigs, or horses), or zoological animals (e.g., monkeys). In particularembodiments, the subject is a human.

While certain embodiments of this invention embrace in vivoapplications, in vitro use of agents of the invention are alsocontemplated for examining the effects of UBE1L and/or ISG15 activationor UBP43 inhibition on particular cells, tissues or regions. In additionto treatment, agents of the invention also find application inmonitoring the phenotypic consequences of enhancing pro-apoptotic anddegradative pathways of neoplastic or pre-neoplastic cells in rodentmodels of cancer.

Agents of the present invention are preferably administered in the formof a pharmaceutical composition which includes the agent in admixturewith a pharmaceutically acceptable vehicle. Desirably, thepharmaceutically acceptable vehicle is selected routinely by those ofskill in the art based upon the type of cancer being treated and theroute of administration best suited for treatment of that type ofcancer. Effective amounts of the agent to be administered can bedetermined routinely by those of skill in the art based upon in vitroand in vivo assays demonstrative of pharmacological activity such asthose described herein.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 Materials and Methods

Cell Culture and Induction Protocol. RA and dimethyl sulfoxide (DMSO)were purchased from Sigma Chemical Company (St. Louis, Mo.). Stock RA(10 mM) solutions were dissolved in DMSO, stored in liquid nitrogen, andused in the dark during experiments. RPMI-1640 and DMEM were purchasedfrom Cellgro/Mediatech (Herndon, Va.).

The NB4 APL cell line expresses PML/RARα (Lanotte, et al. (1991) Blood77:1080-1086). NB4-S1 and NB4-R1 are RA-sensitive and RA-resistantclones of NB4 cells, respectively (Nason-Burchanel, et al. (1997)Differentiation 61:321-331). These cells were cultured in RPMI-1640medium supplemented with 10% FBS (see, Nason-Burchenal et al. (1997)supra).

Chinese hamster ovary (CHO) cells were cultured in DMEM supplementedwith 5% FBS, 100 units/ml penicillin, 100 units/ml streptomycin, and 2mM L-glutamine in a 5% CO₂ humidified incubator at 37° C.

Human bronchial epithelial cells (BEAS-2B) were cultured in serum-freeLHC-9 medium (Biofluids, Rockville, Md.) in accordance with establishedtechniques (Langenfeld, et al. (1997) Proc. Natl. Acad. Sci. USA94:12070-12074; Boyle, et al. (1999) J. Natl. Cancer Inst. 91:373-379).

HeLa cells were cultured in DMEM supplemented with 10% FBS, 100 units/mlpenicillin, 100 units/ml streptomycin, and 2 mM L-glutamine in a 5% CO₂,humidified incubator at 37° C.

The H358 lung cancer cell line was cultured in RPMI-1640 medium(INVITROGEN Corporation, Carlsbad, Calif.) containing L-glutamine, 10%fetal bovine serum and 1% antibiotic-antimycotic solution (CELLGRO,Herndon, Va.) (Petty, et al. (2004) Clin. Cancer Res. 10:7547-54). Cellswere incubated at 37° C. in a humidified incubator with 5% CO₂.Bexarotene treatments of HBE and lung cancer cells were accomplishedaccording to known methods (Dragnev, et al. (2007) Clin. Cancer Res.13:1794-800; Dragnev, et al. (2005) J. Clin. Oncol. 23:8757-64).

Differentiation and Apoptosis Markers. NB4 cell differentiation wasscored by using the nitroblue tetrazolium (NBT) reduction assay(Nason-Burchenal, et al. (1997) supra; Nason-Burchenal, et al. (1998a)Blood 92:1758-1767; Nason-Burchanel, et al. (1998b) Oncogene17:1759-1768) Transductants were identified by green fluorescent protein(GFP) coexpression. Apoptosis was scored by using established techniquesand Hoechst staining of transductants that co-expressed GFP(Nason-Burchenal, et al. (1998a) supra; Nason-Burchenal, et al. (1998b)supra; Stadheim, et al. (2001) Cancer Res. 61:1533-1540). Digital imageswere collected using an OLYMPUS 1X70 inverted microscope, a cooledcharge-coupled device camera, and a MIRACAL Pro Single Cell ImagingSystem (OLYMPUS LSR Research, Melville, N.Y.).

Plasmid Constructs and Transient Transfection. A full length UBE1L cDNAcontaining plasmid was obtained in accordance with the method of Kote,et al. ((1995) Gene Expression 4:163-175). The pGEM-HA-1E1 plasmid wasobtained in accordance with the method of Handley et al. ((1991) Proc.Natl. Acad. Sci. USA 88:250-262). pSG5-HA-1E1 was constructed by cloningthe HA-1E1 fragmented into the pSG5 expression vector. An EcoRI fragmentcontaining the UBE1L cDNA was cloned into EcoRI-restricted pSG5 to yieldthe pSG5-UBE1L plasmid. A truncated UBE1L plasmid (UBE1L-T) lacked anEclXI/SnaBI fragment in the carboxy terminus of UBE1L. The hemagglutinin(HA)-tagged PML/RARα expression vector was constructed frompCMX-PML/RARα and pCMV-HA (CLONTECH) plasmids. The pGL3-UBE1L Lucreporter plasmid contained the luciferase gene and 51 promoter elementsof UBE1L. It was constructed by using a PCR-amplified fragment of theUBE1L promoter derived from NB4-S1 genomic DNA (forward primer, 5′-GCAACC GAG TGA GAC TGT CT-3′, SEQ ID NO:4; reverse primer, 5′-GCG CTC AGAGAT AGG GTT T-3′, SEQ ID NO:5). DNA sequence analysis confirmed thiscloning.

The pcDNA3-UbCH8 plasmid is known in the art (Zhao, et al. (2005) Proc.Natl. Acad. Sci. USA. 102(29):10200-10205). His6-tagged pcDNA3-ISG15 andpcDNA3-His-UBP43 expression vectors are also known in the art(Pitha-Rowe, et al. (2004) J. Biol. Chem. 279: 18178-87; Liu, et al.(2003) J. Biol. Chem. 278:1594-602; Shah, et al. (2008) Mol. Cancer.Ther. 7:905-14). The HA-tagged pRcCMV-cyclin D1 plasmid is known in theart (Rao, et al. (1994) Mol Cell Biol. 14:5259-5267). The lysine-lessHA-tagged cyclin D1 species was previously described (Feng, et al.(2007) Oncogene 26:5098-106). Transient transfection of BEAS-2B cellswas accomplished using EFFECTENE transfection reagent (QIAGEN, Valencia,Calif.) and optimized methods (Feng, et al. (2007) supra). Transfectionsof pSG5-UBE1L, insertless pSG5 vector or with small interfering RNAs(siRNAs) were accomplished using established techniques (Pitha-Rowe, etal. (2004) Cancer Res. 64:8109-15; Shah, et al. (2008) supra). Transienttransfection of BEAS-2B or CHO cells was accomplished by using EFFECTENEand transfection methods in accordance with the manufacturer'sinstructions (Qiagen, Valencia, Calif.). A β-galactosidase reporterplasmid (pCH111) was cotransfected to control for transfectionefficiencies.

SiRNAs targeting UBE1L or a RISC-free control siRNA were synthesized(Dharmacon, Lafayette, Colo.). Different siRNAs were designed to targetUBE1L: UBE1L siRNA1 (5′-CGA CAA CTT TCT CCC GTT A-3′, SEQ ID NO:6) andsiRNA2 (5′-CCT CGG AGT TAG GGC GAA T-3′, SEQ ID NO:7). Transfectionefficiency was monitored by transfecting SIGLO Green TransfectionIndicator (Dharmacon). Percent of cells transfected was assayed by flowcytometry.

UBE1L mRNA Expression Assays. UBE1L mRNA expression was assessed by areverse transcription-PCR assay in accordance with established methods(Kakizuka et al. (1991) Cell 68:663-674). The forward primer was 5′-AGGTGG CCA AGA ACT TGG TT-3′ (SEQ ID NO:8), and the reverse primer was5′-CAC CAC CTG GAA GTC CAA CA-3′ (SEQ ID NO:9). The PCR product wasvisualized by probing with a ³²P-labeled primer. Results were confirmedindependently by Northern analysis using a 1.0-kbEcoRI/NcoI-radiolabeled UBE1L probe in accordance with standardtechniques (Langenfeld et al. (1997) Proc. Natl. Acad. Sci. USA94:12070-12074). This probe had limited homology to E1.

Generation of Anti-UBE1L Antisera. Two rabbit polyclonal antibodiesagainst UBE1L were independently derived (Covance Research Products,Denver, Pa.) using one peptide within the amino terminus(Asp-Cys-Asp-Pro-Arg-Ser-Ile-His-Val-Arg-Glu-Asp-Gly-Ser-Leu-Glu-Ile-Gly-Asp(SEQ ID NO:10)) and a second peptide within the carboxyl terminus(Pro-Gly-Ser-Gln-Asp-Trp-Thr-Ala-Leu-Arg-Glu-Leu-Leu-Lys-Leu-Leu (SEQ IDNO:11)). Specificities of these antisera were confirmed by immunoblotanalyses of UBE1L-transfected CHO cells.

Immunoblot Analysis. Immunoblot analyses were performed usingestablished techniques (Langenfeld, et al. (1997) Proc. Natl. Acad. Sci.USA 94:12070-12074; Spinella, et al. (1999) J. Biol. Chem.274:22013-22018). Anti-RARα antibody was provided by and can bepurchased from P. Chambon (Institut National de la Sant et de laRecherche Mdicale, Strasbourg, France) to detect PML/RARα(Nason-Burchenal, et al. (1998) Blood 92:1758-1767; Nason-Burchenal, etal. (1998) Oncogene 17:1759-1768). An anti-HA mAb was purchased (Babco,Richmond, Calif.) as was an anti-actin polyclonal antibody, C-11 (SantaCruz Biotechnology, Santa Cruz, Calif.).

Cells were lysed with ice-cold radioimmunoprecipitation (RIPA) lysisbuffer, using established techniques (Pitha-Rowe, et al. (2004) supra;Pitha-Rowe, et al. (2004) supra; Feng, et al. (2007) supra; Ma, et al.(2005) Cancer Res. 65:6476-83). Lysates were size-fractionated by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assaysbefore transfer to nitrocellulose membranes (Schleicher & SchuellBioscience, Inc., Keene, N.H.). The polyclonal antibody recognizing theUBE1L amino terminus of UBE1L was used for immunoblot andimmunohistochemical assays. Other primary antibodies for immunoblotassays included a rabbit polyclonal antibody recognizing cyclin D1(M-20) (Santa Cruz Biotechnology, Santa Cruz, Calif.), a murinemonoclonal antibody against hemagglutinin (HA)-tagged proteins (Babco,Richmond, Calif.) and a goat polyclonal antibody recognizing actin(Santa Cruz Biotechnology). Anti-mouse and anti-rabbit antisera werepurchased (Amersham Biosciences, Piscataway, N.J.) as was anti-goatantisera (Santa Cruz Biotechnology) and these were used as respectivesecondary antibodies. Membranes used for immunoblot analyses weretreated with the MEMCODE reversible stain (Pierce, Rockford, Ill.).Treatment with the proteasome inhibitor ALLN was used (Ma, et al. (2005)supra). Quantification of signal intensities was scored as before (Shah,et al. (2008) supra; Feng, et al. (2007) supra; Ma, et al. (2005)supra). To assess cyclin D1 protein stability following UBE1Ltransfection, cells were treated with or without CHX (40 μg/ml) (Feng,et al. (2007) supra).

Retroviral Constructs and Transduction Procedures. MSCV-IRES-GFP wasconstructed to express UBE1L cDNA by cloning an EcoRI fragment frompSG5-UBE1L into an EcoRI site of this retroviral vector. Restrictionendonuclease and partial DNA sequence analyses confirmed cloning was inthe desired orientation. A vector without an insert served as a control.For each vector, 10 μg was transiently transfected using calciumphosphate precipitation along with the CELLPHECT Transfection kit(Amersham Pharmacia, Piscataway, N.J.). The 293GPG packaging cell linewas obtained from Harvard University (Cambridge, Mass.) and is availableto other investigators. Forty-eight hours later viral supernatant from293GPG transfectants (Ory, et al. (1996) Proc. Natl. Acad. Sci. USA93:11400-11406) was used to transduce NB4-S1 or HeLa cells in thepresence of 6 μg/ml POLYBRENE (Sigma Chemical Company, St. Louis, Mo.).Twenty-four hours later, FACS analysis was performed, and cells positivefor GFP expression were harvested by sorting and used for theseexperiments.

Five μg of MSCVIRES-UBE1L-GFP and an empty vector were independentlytransfected into the Phoenix Amphotropic packaging cell line (ATCC,Manassas, Va.) using FUGENE 6 (Roche, Indianapolis, Ind.), and thevendor's recommended procedures. Forty-eight hours later, viralsupernatants were harvested from respective transduced BEAS-2B HBE orH358 lung cancer cells with 4 μg/ml POLYBRENE (Sigma). Forty-eight hourslater, cells positive for GFP expression were harvested using a FACSTARPlus (Becton Dickinson, San Jose, Calif.) high speed sorting cytometer.This was repeated one week later to enrich for studied transductants.

Immunoprecipitation and Pull-Down Assays. After BEAS-2B cells weretransiently transfected with indicated expression vectors, transfectantswere lysed with RIPA buffer for immunoprecipitation or forNi-NTA-agarose (INVITROGEN) pulldown using optimized procedures (Shah,et al. (2008) supra). Anti-HA antibody (Santa Cruz Biotechnology) andprotein A/G beads (Santa Cruz Biotechnology) were used. Ni-NTA-agarosepull-down assays were performed according to conventional methods (see,e.g., Shah, et al. (2008) supra).

Translational Research Studies. Paraffin-embedded and formalin-fixedtissues were obtained from an Institutional Review Board-approved proofof principle bexarotene lung cancer trial (Dragnev, et al. (2007)supra). Tissues were examined for cyclin D1 (Petty, et al. (2004) supra;Dragnev, et al. (2007) supra; Dragnev, et al. (2005) supra), UBE1L(Pitha-Rowe, et al. (2004) supra), ISG15 (Andersen, et al. (2006) Br. J.Cancer 94:1465-71), and Ki-67 (Petty, et al. (2004) supra; Dragnev, etal. (2007) supra; Dragnev, et al. (2005) supra) immunohistochemicalexpression profiles.

Clonal Growth Assays. Clonal growth assays (Langenfeld, et al. (1996)Oncogene 13:1983-90) were performed using 5×10² BEAS-2B and 1×10³ H358cells. These cells were independently engineered to over-express UBE1Lor a control vector. Colonies were treated with bexarotene or vehicle(dimethyl sulfoxide, DMSO) to determine dose-responsive effects. Twoweeks later, visible colonies were fixed and stained with DIFF QUIKsolution (Baxtor, McGaw Park, Ill.) and quantified with the Col Countinstrument (Oxford Optronix, Oxford, UK).

EXAMPLE 2 Role of UBE1L and the UBE1L-Dependent Pathway in APL

All-trans-retinoic acid (RA) treatment induces remissions in acutepromyelocytic leukemia (APL) cases expressing the t(15; 17) geneproduct, promyelocytic leukemia (PML)/retinoic acid receptors(PML/RARα). Microarray analyses have revealed induction of UBE1L(ubiquitin-activating enzyme E1-like) after RA treatment of NB4 APLcells. The kinetics of this induction was studied in RA-sensitive NB4-S1APL cells using a reverse transcription-PCR assay. UBE1L mRNA inductionoccurred by three hours after 10 μM RA treatment. These results wereindependently confirmed by northern analysis and after 1 μM RA treatmentby reverse transcription-PCR assay. In contrast, UBE1L expression wasnot induced during the same time period, despite 10 μM RA treatment ofthe RA-resistant NB4-R1 cell line. This is indicative of UBE1L being adirect retinoid target.

Further, the direct relationship between UBE1L induction and effectiveretinoid treatment of APL cells was demonstrated by examination of UBE1Limmunoblot expression. For these experiments, immunogenic peptidesdescribed herein were used to generate independent polyclonal antiserarecognizing the amino or carboxyl termini of UBE1L protein,respectively. Chinese Hamster Ovary (CHO) cells that did not basallyexpress UBE1L mRNA were transfected with a full-length UBE1L cDNA or aninsertless vector. CHO cells transfected with UBE1L expressed UBE1Lprotein. In contrast, cells transfected with an insertless vector didnot express this 112-kDa species. The UBE1L immunoblot expressionprofiles were also compared in RA-sensitive versus RA-resistant NB4cells. UBE1L protein was basally expressed at low levels in both celllines. However, protein expression was induced only after RA (1 μM)treatment of NB4-S1 APL cells.

A hallmark of RA response in APL is PML/RARα degradation (Yoshida, etal. (1996) supra; Raelson, et al. (1996) supra; Nervi, et al. (1998)supra; Zhu, et al. (1999)). RA treatment has been reported to repressPML/RARα expression in NB4-S1, but not in RA-resistant NB4-R1 cells(Nason-Burchenal (1997) Differentiation 61:321-331). To examine therelationship in APL cells between UBE1L and PML/RARα expression,immunoblot expression profiles for these species were examined beforeand after 24 hour RA (1 μM) treatment of NB4-S1 cells. An inverserelationship was evident between UBE1L and PML/RARα expression bothbefore and after RA treatment thus indicating a direct role for PML/RARαin regulating UBE1L expression.

A 1.3-kb fragment of the UBE1L promoter was then demonstrated to becapable of mediating transcriptional response to RA in a retinoidreceptor-selective manner. PML/RARα, a repressor of RA target genes,abolished this UBE1L promoter activity. To examine the potential forPML/RARα to affect UBE1L, 1.3 kilobases (kb) of the UBE1L promoterupstream of the ATG translation start site was cloned into aluciferase-containing reporter plasmid. This reporter plasmid wastransfected into CHO cells in the presence and absence of RA treatment.This fragment of the UBE1L promoter was capable of mediatingtranscriptional response to RA in a retinoid receptor-selective manner.

The relationship between PML/RARα and activity of this UBE1L reporterplasmid was examined when PML/RARα was cotransfected with this reporterplasmid. Cotransfection of PML/RARα with RARE led to a marked repressionof UBE1L reporter activity before and after RA (1 μM) treatment. Thisinhibition depended on the dosage of transfected PML/RARα. In eachexperiment, a cotransfected β-galactosidase reporter plasmid was used tocontrol for transfection efficiencies. No appreciable effect of PML/RARαon the transcriptional activity of the β-galactosidase reporter plasmidwas observed. Thus, PML/RARα repressed activity of this UBE1L reporterplasmid.

UBE1L has homology to E1. However, E1 mRNA was not induced after RA (1μM) treatment of NB4-S1 cells, thus indicating different effects of RAon expression of UBE1L and E1.

A hallmark of RA response in APL is PML/RARα degradation. Accordingly,the ability of UBE1L as well as E1 to trigger PML/RARα degradation wasnext examined. Cotransfection assays were performed using cells that donot express PML/RARα. In these experiments CHO cells that did notexpress UBE1L and BEAS-2B cells that expressed low levels of UBE1L, butcould be readily transfected with RARs or PML/RARα, were used.Degradation of transfected PML/RARα was triggered by UBE1L in adose-dependent manner after transfection of CHO cells or BEAS-2B cells.This degradation of PML/RARα occurred in the absence of RA treatment.The PML domain of PML/RARα appeared to be more sensitive to degradationby UBE1L than the RARE domain. Transfection of a truncated UBE1L(pSG5-UBE1L-T) did not cause PML/RARα degradation. To establish thatPML/RARα degradation was a distinct UBE1L function, E1 was alsotransfected into BEAS-2B cells with PML/RARα E1 did not cause PML/RARαdegradation. Thus, transfection of UBE1L, but not E1, led to PML/RARαdegradation even without RA treatment.

The effects of engineered overexpression of UBE1L in APL cells on growthor differentiation state of these cells was then examined. Tooverexpress UBE1L in APL cells, retroviral vectors (Ory, et al. (1996)supra) were constructed to express UBE1L or no insert. Coexpressed GFPwas used to enrich for retroviral-expressing cells after FACS sorting.HeLa cells, which do not express PML/RARα and basally express UBE1L atlow levels, were used as a control for these experiments becauseretroviral transduction conditions were previously optimized in thesecells. UBE1L overexpression was engineered independently in NB4-S1 andHeLa cells using the described retroviral transduction method. As acontrol, an insertless control vector was independently introduced intothese cell lines as confirmed by immunoblot analysis. A strikingdifference in biological effects was observed after transduction ofUBE1L into NB4-S1 versus HeLa cells. UBE1L overexpression in NB4-S1cells resulted in the rapid induction of apoptosis as measured by theHoechst staining of transduced cells. Three independent fields wereexamined for the insertless control NB4-S1 transfectants and 5.1% ofthese cells were apoptotic. Analysis of UBE1L-transduced NB4-S1 cellsrevealed a high proportion (39.7%) of apoptotic cells. Thesetransductants did not exhibit morphological evidence of leukemic cellmaturation. This lack of induced differentiation was confirmed by theabsence of NET-positive cells (see Table 1).

TABLE 1 Cell line NBT, % NB4-S1 (−RA) 0 NB4-S1 (+RA) 92 NB4-S1 (−UBE1L)0 NB4-S1 (+UBE1L) 0 NBT maturation assays performed on NB4-S1 APL cellsafter 5 days treatment with RA (1 μM) [designated +RA} or vehicle (DMSO[designated −RA] or transduction of the UBE1L retrovirus, [designated+UBE1L] as compared to transduction of the same retrovirus without aninsert (designated −UBE1L].

Promotion of apoptosis was not observed in HeLa cells transduced witheither the UBE1L or insertless retroviral vector. Thus, UBE1Ltransduction preferentially triggered apoptosis in PML/RARα-expressingcells. Induction of apoptosis was so rapid, however, that examination ofthe mechanisms signaling apoptosis was precluded.

As demonstrated herein, there is a tight link between UBE1L inductionand PML/RARα degradation. More specifically, experiments describedherein are demonstrative of an antagonistic relationship between UBE1Land PML/RARα. Further, increases in expression of UBE1L rapidly induceapoptosis in cells expressing PML/RARα. Accordingly, UBE1L is believedto be a target for repression by PML/RARα and induction of apoptosis incells expressing this oncogenic protein.

Additional experiments in BEAS-2B human bronchial epithelial cellsconfirmed that co-transfection of UBE1L with transfected cyclin D1triggers degradation of the oncogenic protein cyclin D1. Thus, thedegradation program described herein is active beyond leukemia. In thisstudy β-actin was used as a control to confirm that similar amounts oftotal protein were added per lane. Prior work has implicated cyclin D1degradation as a chemopreventive target (Langenfeld, et al. (1997)supra; Boyle, et al. (1999) supra). Thus, these experiments directlyimplicate UBE1L in cancer chemoprevention. The deconjugase UBP43 is alsoimplicated in the described degradation program in that transfection ofUBP43 stabilizes cyclin D1 despite transfection of UDE1L in BEAS-2Bhuman bronchial epithelial cells.

RA also augments the ubiquitin-like protein ISG15 expression inRA-sensitive but not resistant NB4 cells. In addition, RA treatmentincreases intracellular ISG15 conjugation in retinoid-sensitive NB4cells. This is indicative of a link between increased ISG15 and UBE1Lexpression and induction of myeloid differentiation. Consistent withthis is that RA treatment increases intracellular ISG15 conjugation inretinoid-sensitive NB4 cells. A physical interaction between UBE1L andISG15 was established in vivo using a transient co-transfection assay.This interaction was not observed when mutant ISG15 lacking essentialC-terminal glycines was examined. Thus, these experiments are indicativeof a coordinate regulation of UBE1L and ISG15. PML/RARα degradation wasalso shown to be inhibited by UBP43 transfection as confirmed byco-transfection experiments with PML/RARα and UBP43 where UBP43 was ableto overcome the ability of UBE1L to trigger degradation of PML/RARα.Accordingly, it is contemplated that, like UBE1L, induction of ISG15 orinhibition of UBP43 can also enhance degradation of oncogenic proteinssuch as PML/RARα and promote apoptosis of cancer cells expressing theseoncogenic proteins.

EXAMPLE 3 UBE1L Causes Lung Cancer Growth Suppression by TargetingCyclin D1

UBE1L is implicated as a molecular pharmacologic target inhibitingcyclin D1. This has provided a mechanism for the tumor suppressive roleof UBE1L. To determine whether UBE1L affects cyclin expression, BEAS-2Bcells were co-transfected with UBE1L and independently with cyclin D1,cyclin D2, cyclin D3 or cyclin E. Only cyclin D1 was inhibited by UBE1Land actin expression was unaffected.

Immunoblot experiments were conducted following transfection of UBP43,the enzyme leading to ISG15 deconjugation. Dose-dependent effects inBEAS-2B cells of transient UBP43 transfection were observed on cyclin D1protein. Cyclin D1 expression increased as UBP43 transfection dosageincreased. UBE1L transfection inhibited cyclin D1 expression in BEAS-2Bcells, but UBP43 co-transfection antagonized this effect (FIG. 1). Toestablish UBE1L affected cyclin D1 protein stability, UBE1L wasco-transfected with HA-tagged cyclin D1 into BEAS-2B cells in thepresence and absence of CHX. This analysis revealed that UBE1L reducedexogenous cyclin D1 protein stability following CHX treatment. UBE1Ltransfection also reduced endogenous cyclin D1 protein, but not cyclinD1 mRNA expression in BEAS-2B cells.

It was contemplated that the ubiquitin-like protein ISG15 would complexwith cyclin D1. BEAS-2B cells were transiently transfected with orwithout cyclin D1 and with or without UBE1L and ISG15 expressionvectors. Lysates were subjected to immunoprecipitation before immunoblotanalyses. These analyses revealed two major conjugates of cyclin D1following co-transfection of UBE1L, ISG15 and cyclin D1. Transfection ofUbCH8, the E2 enzyme for ISG15ylation (Zhao, et al. (2004) Proc. Natl.Acad. Sci. USA 101:7578-82) did not appreciably change this conjugation,indicating endogenous UbCH8 expression was not limiting. Treatment withthe proteasome inhibitor ALLN stabilized ISG15ylated species. TheseISG15-conjugated cyclin D1 species were reduced by UBP43co-transfection. Indeed, UBP43 over-expression promoted cell growth, butUBP43 knock-down (by small interfering RNA (siRNA) and by short hairpinRNA (shRNA)-based approaches) repressed growth and induced apoptosis.

To establish UBP43 as a molecular pharmacologic target of generalimportance in cancer therapy or cancer prevention, unique polyclonalantibodies against, respectively, the amino and the carboxyl termini ofUBP43 were developed and used in an immunoblot assay for UBP43detection. Results revealed abundant UBP43 expression in all cancer celllines examined (including lung cancer and APL). Findings were extendedby use of these antibodies for immunohistochemical assays onparaffin-embedded clinical surgical biopsies that revealed frequentover-expression of UBP43 in human cancers.

Specific lysines in cyclin D1 affect cyclin D1 protein stability (Feng,et al. (2007) supra). Whether a lysine-less cyclin D1 species wasresistant to UBE1L-mediated inhibition of cyclin D1 was studied andfound. This was an expected outcome since absence of lysine residuesprevented ISG15ylation. To confirm this inhibition involved a complexbetween ISG15 and cyclin D1, transfected lysine-less cyclin D1 wasimmunoprecipitated with anti-HA or pulled-down with Ni-NTA,respectively, before immunoblotting with an anti-HA antibody. Thisanalysis revealed that UBE1L inhibition of cyclin D1 depended onconjugation to lysine(s) within cyclin D1.

Effects of UBE1L expression on BEAS-2B HBE and H358 lung cancer cellgrowth were studied. Retroviral UBE1L expression was independentlyaccomplished in BEAS-2B and H358 cells. Exogenous UBE1L reducedendogenous cyclin D1 expression in both transduced cell lines (relativeto controls). A limiting dilution clonal growth assay confirmed UBE1Lover-expression conferred a marked repression of clonal growth. Similarresults were obtained in replicate experiments. BEAS-2B and H358 UBE1Ltransductants exhibited at least a 50% repression (P<0.001) of clonalgrowth versus insertless controls. To determine whether knock-down ofUBE1L affected cyclin D1 expression, two independent siRNAs targetingUBE1L and a control siRNA were independently transfected into ED-1murine lung cancer cells, which exhibit high basal UBE1L proteinexpression. Over 90% of these cells were transiently transfected.Knock-down of UBE1L by these siRNAs increased cyclin D1 immunoblotexpression in ED-1 cells.

Prolonged RA-treatment augments UBE1L expression in BEAS-2B cells(Pitha-Rowe, et al. (2004) Cancer Res. 64:8109-15). Studies wereundertaken to assess UBE1L expression after treatment of BEAS-2B andH358 cells with bexaratene, a retinoid X receptor agonist. Bexarotenerepressed cyclin D1 protein in BEAS-2B and H358 cells. Bexarotene (1 μM)prominently increased UBE1L expression as determined by immunoblotanalysis following 10 days treatment of BEAS-2B cells versus vehicletreatment. Similar findings were obtained in H358. Alpha-actinexpression served as loading control. Bexarotene treatment also caused adose-dependent decline of BEAS-2B clonal growth (FIG. 2).

Cyclin D1 immunohistochemical expression declines when high bexarotenelevels are measured in lung tumors (Dragnev, et al. (2007) Clin. CancerRes. 13:1794-800). Whether this repression occurred with an increasedUBE1L expression in bexarotene post-versus pretreatment lung cancerbiopsies was studied. ISG15 immunohistochemical expression was similarin post- and pre-bexarotene treatment biopsies of the lung cancer cases.In contrast, cyclin D1 expression declined in post-versus pre-treatmentbiopsies when high bexarotene plasma (1.49 μM) and intratumoral (0.31μM) levels were measured (Dragnev, et al. (2007) supra). Upon furtheranalysis of this case, UBE1L immunohistochemical expression was shown toincrease with bexarotene treatment and proliferation decrease inpost-versus pretreatment biopsies, as assessed by Ki-67 immunostaining.

Another representative case was examined. This case had low plasma (0.13μM) and intratumoral (0.09 μM) bexarotene levels (Dragnev, et al. (2007)supra). UBE1L and cyclin D1 immunohistochemical expression profiles werenot L appreciably altered by bexarotene treatment. Repression of Ki-67immunostaining was not observed. A total of five cases were examinedwith only one having high intratumoral bexarotene levels and alsoregulation of UBE1L, cyclin D1 and Ki-67 expression. The response ratefor UBE1L was 20% (95% CI 1, 72). The odds ratio (OR) assessingassociation between intratumoral bexarotene concentration and UBE1Lincrease was 6 (95% asymptotic CI) (0.1, 354.9). This OR indicates ahigh probability of UBE1L induction in tumors with high bexarotenecompared to tumors with low bexarotene levels.

1. A method for enhancing pro-apoptotic and degradative pathways ofneoplastic or pre-neoplastic cells comprising contacting cells with anagent that selectively induces UBE1L or a ubiquitin-like protein ISG15,or selectively inhibits the deconjugase UBP43.
 2. A method forpreventing or treating cancer in a subject comprising administering to asubject in need of treatment an effective amount of an agent thatselectively induces UBE1L or a ubiquitin-like protein ISG15, orselectively inhibits the deconjugase UBP43 thereby preventing ortreating cancer in the subject.
 3. A method for identifying an agent foruse in preventing or treating cancer comprising determining an agent'sability to induce UBE1L or ubiquitin-like protein ISG15, or to inhibitdeconjugase UBP43 thereby identifying an agent for use in preventing ortreating cancer.
 4. An agent identified by the method of claim 3.